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THE OURNAL F BIOLOGICALHEMISTRYVol. 257, No. 22, Issue of November 25. pp. 13246-13252,1982Printed in U.S.A.

Regulation of the Cytoplasmic pH in Streptococcus faecaZis*(Received for publication, June 1 1982)

Hiroshi KobayashiS, Naoto Murakami, and TsutomuUnemotoFrom the DeDartment o Membrane Biochernistrv. Research Institute forChemobiodynamics, Chiba University, 1-8-1,

the cytoplasmic pH. One is tha t thecytoplasmic pH is raisedby metabolic extrusion of protons and electrogenic influx ofpotassium when bacteria are growing on the acid medium 5-10).The other s tha t sodium proton antiporter orpotassiumproton antiporter lowers the cytoplasmic pH when bacteriaare growing on the alkaline medium (5, 11-20).

Thus, these studies indicate tha t the cytoplasmic pH isregulated by the transport ystems for cations. However, thedetailed mechanism is still unclear. How do bacteriause thesetransport systems to regulate the cytoplasmic pH? For themaintenance of the cytoplasmic pH ateutrality, it isessentialtha t the ctivity of some of the transport ystemsis regulatedby the cytoplasmic pH. Which one is such a system? Thesepoints are unclear.

We have studied he regulatory mechanism of the cytoplas-mic pH in Streptococcus faecalis. S. faecalis has no respira-tory chain and protons expelled by the proton-translocatingATPase (H+-ATPase)which is essentially the same enzymeas the coupling factor of oxidative phosphorylation (6, 7). Inthis paper, we propose the regulatory mechanism of the cy-toplasmic pH in S. faecalis. Th e essential points of it are thefollowing: 1) S. faecalis has the machinery to raise the cyto-plasmic pH but not one to lower it, 2) the cytoplasm isalkalized by an extrusion of protons mediated by the H-ATPase and by an influx of cations, and 3) the activity of theH+-ATPase s very low at pH above8.0 and consequently thecytoplasmic pH is maintained a t near 8.0.

MATERIALSANDMETHODS

Znohana, Chiba, 280, Jhpan -

Streptococcusfaecalis grows at optimum rate when agrowth medium is at pH 6.5-8.0. Within this range ofpH, the cytoplasmic pH is regulated at near 8.0. Theregulatory mechanismof the cytoplasmic pH has beeninvestigated in S. f a e c a l i s . Under normal conditions,the cytoplasmic pH is regulated by an extrusion ofprotons mediated by the proton-translocating ATPase(H+-ATPase) and by an influx of K+ mediated by thetransport systems for + n S. faecalis.The cytoplasmicpH can be regulated normally withan influx of K+ viavalinomycin. Mutant 7683 which is defective in heextrusion system forNa+ is able to accumulate Na+ viaa leak pathway in the presence of the protonmotiveforce (Heefner, D. L., and Harold, F. M. 1980)J.Biol.Chem. 255, 11396-11402).he cytoplasmic pH of thismutant is also regulated at near 8.0 with the influx ofNa+ via the leak pathway. These results suggest tha tthe cytoplasmic pH is normally regulated even if theinflux of cations is not mediatedy a specific tran spor tsystem. Furthermore, it is suggested that S. faecalishas no system depending onhe cellular metabolism ofenergy for the cidification of the cytoplasm. The gen-eration of the protonmotive force drastically decreaseswhen th e cytoplasmic pH is above 8.0 and ATP hydrol-ysis activity of the H+-ATPase is very lowat pH above8.0. From these results,we propose the following reg-ulatory mechanism of the cytoplasmic pH: th e cyto-plasm is alkalized by the proton extrusionvia the H+-ATPase and by the cation influx, and the activ ityf thealkalization decreases by the decline of the act ivity ofthe H+-ATPase when the cytoplasmic pH approaches8.0. Thus, the cytoplasmic pH is regulated by the H+-ATPase itself.

It isnow generally accepted tha t many of bacterial transportsystems are linked to the irculation of proton ( 1 , 2 ) .Recently,it has been pointed out that ion circulation is not obligatoryfor bacterial growth (3, 4). f it is true, why do bacteria needmany kinds of the transport ystems for ions and metabolites?We think one of the answers is that transport systems arerequired for an environmental adapta tion of bacteria: theoptimum conditions of bacterial cytoplasm are maintained bythe aid of the ransport systems only when bacteria aregrowing in harsh environments.

Various reports (for review, see Ref. 5) have revealed thatthe cytoplasmic pH is near neutrality in all bacteria studiedeven if they are growing in acid or alkaline medium. Therehave been two lines of reports to ccount for thi s constancy of

The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.To whom all the correspondence should be addressed.

Organisms and Growth Media-Streptococcus faecalis (ATCC9790, wild type), mutan t7683 (defective in sodium transport syst em),and mutant 687A (defective in retention of cellular potassium) weregenerously supplied by Dr. F. M. Harold, National Jewish Hospitaland Research Center, Denver. Mutant AS25 was derived from S.faecalis 9790 as described previously (21). Bacteria were grown over-night at 37 Con he following complex media.Medium KTYcontained 10 g of K,HP04, 10 g of tryptone, 5 g of yeast extract, and10 g of glucose per liter. Medium 2KTY contained 20 g of KzHP04instead of 10 g. Medium NaTY contained 8.5 g of NaaHPOl insteadof K2HP04.Arginine-adapted cells were grown on the medium con-taining 10 g of K2HP04,10 g of tryptone, 5 g of yeast extract, 10 g ofarginine, and 1 g of galactose per liter.Prep arat ion of Membrane Vesicles an d Measurement of Fluores-cence-The wild type strain and mutantAS25 were grown overnighton medium KTY and LKTY, respectively. Membrane vesicles wereprepared from these strains as described previously (22). Membranevesicles were suspended n buffer (50 mM Trid male ate , 250 mMsucrose, 5 mM MgS04, pH7.5) a t 0.15-0.20 mg of protein/ml and thechanges in the fluorescence of ANS and quinacrine were measuredwith a fluorescence spectrophotometer (Hitachi , MPF4) as escribedpreviously (22).Measurement of the Membrane Potentialn d p H Gradient-The

Th e abbreviations used are: ANS, 1-aniljno-8-naphthalenesulfon-ate; TPMP+, riphenylmethylphosphonium on; TCS, tetrachlorosal-icylanilide; DCCD, N,N-dicyclohexylcarbodiimide; Tricine, N -[tris hydroxymethyl)methyl]glycine.13246


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Regulation of BacterialytoplasmicH 13247membrane potential and thepH gradient (inside minus outside)weremeasured with [methyl-H]TPMP+ and [carb~xyl-~C]acetylsalicylicacid, respectively. Cells washed with2 mM MgS0, were suspended inthe buffer indicated. [carbo~yl-~C]Acetylsalicyliccid (10 p ~ 8.3mCi/mmol) was added to the cell suspension and the mixture wasincubated at 25 C.At zero time, 10 mM glucose was addedandaliquots (500 pl) were filtrated through a Millipore filter (pore size,0.45 p ) . After washing of the filters with 2 ml of the same buffer (twotimes), he adioactivityon he ilters was measured in a iquidscintillation counter (Packard, 3255). [14C]Methylamine 11 p ~ ,4mCi/mmol) was also used to measu re the negative pH grad ient . Theprocedure for the measurement of the membrane potential was thesame as thatof the pH gradient except that [methyl-HITPMP 20p ~ ,7 mCi/mmol) and membrane filter (S&SCorp.; pore size, 0.45p ) were used. We measured the radioac tivity of these materials onthe fiiter without the addition of cells and subtracted it from thoseobtained in theresence of cells. Interna l water space as determinedas described previously (21) and the value as 3 pl/mg of cell protein.We should consider the nonspecific adsorption of radioactive ma-terials on the cell surface. After subtraction of the amount adsorbedto the filter, the amoun t of acetylsalicylic acid on the filter was lessthan 1 pmol/mg of protein when the negative pH gradient (interioracid) was above 1.0 unit, under our conditions. If the pH gradient(interior alkaline) is 0.1 pH unit, the amoun t of acetylsalicylic acidtaken up by cells is calculated to be 37.8 pmol/mg of protein. Thus,the amountof acetylsalicylic acidadso rbed to ell surface is negligible.Similarly, the adsorptionf methylamine to ell surface was estimatedto be very low. Th e values of the pH g radient obtained by the flow-dialysis method were identical with hat obtained by the metho ddescribed abovewhen hepHgradient was higher han 1.0 unit,indicating that the loss of the radioac tive materia ls rom cells duringfiltration is negligible. It shou ld be noted thatpH gradient less tha n1.0 unit is hard to be measured by the flow-dialysis methods. Wemeasured the pH gradient several times and the standard error wasfound tobe less than 0.1 pH unit.From heseobservations, weconclude that the erro rf the valuesof the pH gradient obtained hereis within 0.1 pH unit.Assay of K Mouement-Cells washedwith 2 mM MgSO, (twotimes) were suspended in the buffer indicated. Net accumulation ofK was determined with the use of a K-electrode as describedpreviously (23). An influx of K was measured with c2K. After additionof2K at the time indicated, aliquots (500 pl) were filtrated througha Millipore filter (HAWP) and theilters were washed two timeswiththe same buffer. Th e radioactivity on the filters was measured in aliquid scintillation counter (Packard, 3255).Other Procedures-K+-depleted cells were prepared by the trea t-ment with monactin asdescribed previously (23) . Internal concentra-tion of K was determined with an atomic absorpti on spectrophotom-eter (Hitachi Perkin-Elmer 03) after cells were collected on polycar-bonate filters (Nucleopore Corp.) a s described previously 23).Theactivity of ATP hydrolysis was assayed a t 25 C in buffer (50 mMTris/maleate or 9 0 mM Tris /Tricine) as described previously (24) .Each buffer contains 5 mM ATP (Tris salt) and mM MgSO,. Proteinwas determined by the method of Lowry et al. (25) .

Reagents-Weacknowledge thegenerous gifts of the followingreagents:monactinand TCS Dr. F. M. Harold,National JewishHospitalandResearchCenter,Denver)and nigericin (Eli LillyCo.). Valinomycin was purchased from Sigma, The ollowing goodbuffers were purchased from Nakarai Kagaku Co. (Kyoto, Japan):Tris and Tricine. [carboxyZ-4C]Acetylsalicyliccid and [%]methyl-amine were purchased from New England Nuclear Co. and K waspurchased from JapanRadioisotope Association. [rnethyZ-H]TPMpwas synthesized by the method of Hong (26). Other reagents usedwere of analytical grade.

RESULTSThe Protonmotive Force and the Cytoplasmic pH as a

Function of the pH ofhe Medium-The present study beganwith the measurement of the generation of the protonmotiveforce and the cytoplasmic pH over a range of medium pHfrom 5.5-8.6. The results shown in Fig. 1 are essentiallyidentical with that observed in other bacteria 8 , 10, 11, 27,28). A peculiar point shown in Fig. 1 is that the generation ofthe protonmotive force was drastically decreased at pH bove7.5. The same phenomenon was reported in Streptococcuslactis (27). On the other hand, such a drastic decrease of the

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- 5 05 . 0 8 . 0 7 . 0 8 . 0 9.0PHou t

FIG. 1. Components of the protonmotive force and the cy-toplasmic pH as a function of the pH of the medium. Washedcells of the wild type stra in grown on medium KTY were suspendedin buffer indicated a t 0.32 mg of cell protein/ml. After addition of 5mM K(maleate), themembranepotentialandpHgradient weremeasured with TPMP and acetylsalicylic acid as described underMaterials and Methods. Th e cytoplasmic pH was calculated fromthepHgradient. Glucose (10 mM) was added at zero timeandsampling of aliquots was done a t 15-21 min. Buffers used were thefollowing: 50 mM Tris/mal eate, pH 5.5-7.5, and 50 mM Tris/Tricine,pH 8.0 and 8.6. All buffers contained 2 mM MgSO,. Symbols: 0 , otalprotonmotive force; A, membrane potential; 0,H gradient ( -59 XpH gradient); A, cytoplasmic pH (pH,,?).generation of the protonmotive force was not observed inEscherichia coli 8)and Micrococcus lysodeikticus (28). Theother feature is tha t the membrane potential is low as com-pared with tha t in other bacteria. Bakker and Harold (29)have reported tha t S. faecalis can generate the membranepotential of approximately -120 mV in the absence of K + andthe membrane potential decreases to approximately -70 mVby the addition of K+ at pH 7.5. In K+-depleted cells, themembrane potential of over -150 mV is generated withoutthe addition of K (30).Our data agree with the data bservedpreviously.The optimum pH for the growth of S. faecalis was 6.5-8.0(21). Within this range, the cytoplasmic pH was regulated atnear 8.0 (Fig. 1).This agrees with the following observations:the cytoplasmic pH becomes equal to that of the mediumupon the addition of gramicidin D (3) and the growth of S.faecalis under tha t condition requires that the pH of themedium is above 7.5 (3, 21). The growth rate was markedlydecreased at pH above 8.0 in the presence of gramicidin D aswell as in its absence (data not shown). It is therefore con-cluded tha t thecytoplasmic pH should be maintained at near8.0 for the optimum growth of S. fuecalzs.

Regulation of the Cytoplasmic pH Is Conducted by H +-ATPase and the Trk System-The generation of a pH gra-dient is required to maintain the cytoplasmic pH at near 8.0when bacteria are growing a t pH below 8.0. It has een shownthat in S. faecalis a proton-translocating ATPase (H+-ATP-ase) expels protons and generates the pH gradient 6 ,7, 31).We isolated mutants defective in the regulation of the cyto-plasmic pH (21). As reported previously 21),neither a mem-brane potential nor a pH gradient was generated in intac tcells of such mutants and theesion of the mutants s a pointmutation. In the resent study,we investigated the generationof the protonmotive force on membrane vesicles from AS25,one of such mutants.Membrane vesicles were prepared and the eneration of theprotonmotive force upon the addition of AT P was observedas described under Materials and Methods. In membranevesicles from the wild type stra in, an enhancement of the


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13248 Regulation of Bacterialytoplasmic p Hfluorescence of ANS and a quenching of the fluorescence ofquinacrine were observed upon the addition of ATP , indicat-ing tha t these vesicles were able to generate th e protonmotiveforce (Fig. 2). These changes of fluorescence were completelyinhibited by DCCD and ionophores (Fig. 2). On the contrary ,the addition of ATP induced neither the change of the fluo-rescence of ANS nor th at of quinacrine in membrane vesiclesfrom AS25 (Fig.) , indicating the loss of the ability to generatethe protonmotive force. It should be noted tha t the fluores-cence of ANS was enhanced in he membrane vesiclesof AS25when the diffusion gradient ofK' was established in thepresence of valinomycin and membrane vesicles of AS25 canaccumulate Ca2' as well as the esicles of the wild type stra in(data not shown). These results suggest tha t the lesion inAS25 occurs ina gene for the H'-ATPase. In fact, AT Phydrolysis activity in membrane vesicles from AS25 was ap -proximately 30% of that in membrane vesicles from the wildtype strain. We concluded from these results that the H+-ATPase is essential for the regulation of the cytoplasmic pH.

There have een many reports (5-10) that theK transportsystem has an essential role in the generation of th e pHgradient. As shown in Fig. 3A , upon the addition of glucose,the generation of the pH gradient was accompanied with K+accumulation and he alkalization of the cytoplasmic pHdecreased as the rate f K' accumulation declined. The sam eresult was also obtained at pH 7.0 (Fig. 3B ) . In this experi-ment, the pH gradient was negative (interior acid) at zerotime and the cytoplasm became more alkaline than the me-dium after 12 min at pH 6.0 (Fig. 3A ) . We suppose that thenegative gradient of pH at anarly stages due to theollowingfacts: 1) the cytoplasmic pH of cellsgrown overnight onmedium KTY is 5.0 or below and 2) the cytoplasm is moreacid than themedium unless the system to raise he cytoplas-mic pH does function as discussed below.

Mutant 687A, which was originally isolated as a mutantdefective in the retention of the cellular K' by Harold et al.(32),has a reduced activity of an accumulation of K'. In thisstrain, the pH gradient was generated slowly and maximummagnitude of the pH gradient was also small, correspondingwith the reduced activity of K + accumulation (Fig. 3C) .

These results uggest tha t the eneration of the pH gradientis accompanied with the accumulation of K'. S. faecalis hastwo transport systems for K , the Trk and Kdpystems (23).K' accumulation observed above is mediated by the Trk

25 decrease

FIG. 2 . Generationof heprotonmotive orceby ATP inmembrane vesicles fromhe wild type strain and mutantS25.The changes of fluorescence of ANS and quinacrine were measuredfrom mutant AS25 (dotted line) as described under Materials andin membrane vesicles from the wild type strain (solid line)and thatMethods. ATP 1 mM) was added at the arrow. Supplements werepresent as follows:1,none; 2, valinomycin and nigericin, 2 pg/ml each,plus 5 mM KC1; 3, preincubated 15 min with 0.2 mM DCCD.

Time min )FIG. 3. Generation of the pH gradient accompanied withK

uptake. Washed cells of the wild type and 687A strains grown onmedium NaTY were suspended in buffer 50 mM Tris/maleate, 2 mMMgS04, 2 mM KCI) . The pH gradient (solid ine) and K' uptake(dotted line)were measuredas described under Materialsand Meth-ods. Acetylsalicylic acid was used for the measurement of the pHgradient. Glucose (10 mM) was added at zero time. A , wild type, pH6.0 (0.45 mg of cell protein/ml); B, wild type, pH 7.0 (0.43 mg of cellprotein/ml); C, mutant 687A. pH 6.0 (0.27mg of cell protein/ml).

I 1 I l l2.0 A B x

Iime minFIG. . The rate of K + influx. Washed cells of the wild typestrain grown on medium NaTY were suspended in buffer (50 mMTris/maleate, 2 mM MgS04 , 2 mM KC1) at the following pH values:6.0 (A and B ) , 7.0 ( C ) ,8.0 ( D l .42Kwas added at the arrow. Glucose10 mM) was added at zero time. The net uptake (dotted line) andinflux (solid line) of K Cwere measuredas described under Materialsand Methods. Protein concentrations were 0.45 ( Aand B ) ,0.46 ( C ) ,and 0.41 D) g/ml.system because the Kdp system is not expressed under theseconditions ( 23 ) . Rubidium ion is accumulated by theTrksystem of S.faecalis (23) and th e pH gradient was generatedin the presence of Rb' instead of K' (dat a not shown).

An Influx of K s Dependent on the Cytoplasmic pH-Asmentioned above, the generation of a pH gradient is accom-panied with K' accumulation and he alkalization of thecytoplasmic pH decreases as the rat e of K+ accumulationdeclines (Fig. 3). We next observed the influx rate of K' underthe conditions in Fig. 3. An influx rate of K' at the steadystate of K' accumulation was lower than that at the initialstage in the medium at pH 6.0 (Fig. 4,A and B). An effluxrate of K + given n influx rate minus net uptake at thesteady state is close to tha t at the nitial stage (Fig.4, A andB ) , ndicating tha t the decrease of net uptake of K' is mainlydue to the decrease of the influx rate.The influx rate of K' at the steady state level of K'accumulation was higher at pH6.0 than that atpH 7.0 (Fig.4, B and C .Bakker and Harold (29) have proposed tha t, in S.faecalis, K influx is subjected to feedback regulation: a rat eof the influx declines as the cellular level of K' increases. In


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Regulation of Bacterial Cytoplasmic pH 13249contrast to their proposal, intracellular concentrations of K+at the steady state level were 2.9 and 2.4 pmol/mg of proteinat pH 6.0 and 7.0, respectively (Table I). Moreover, the influxrate of K in the medium at pH 8.0 was very close to thatobtained a t pH 7.0 (Fig.40, able I) and the concentration ofcellular K was 1.7 pmol/mg of protein (Table I). We meas-ured the cytoplasmic pH under the same conditions. Evi-dently, the influx rate of K was dependent on the cytoplasmicpH rather than theellular level of K (Table I) . These resultssuggest that the influx rate rather than thenet uptake of Kis important for the generation of .the pH gradient and thedecrease of the influx rate is closely related to thedecrease ofthe generation of the pH gradient.In Fig. 4D, the uptake of K did not reach to the steadystate level at pH 8.0, although the cytoplasmic pH reaches tothe steady stateevel (pH 8.2)within 5 min. The efflux rate ofK (influx rate minus net uptake rate) is negligible at pH 8.0(Fig.4 0 ) . Therefore, it is thought that theefflux system doesnot work and consequently cells continue the net uptake ofK'. In any case, the important point shown in Fig.40 s thatthe influx rate of K' is low when the cytoplasmic pH is 8.2.

Generation of a pH Gradient I s Established by the Influxof Cation viaAny Route Other Than theTrk System-Bakkerand Mangerich (8)have pointed out tha t K+ influx leads to adepolarization which allows more protons to be pumped outand consequently the magnitude of a pH gradient increases.Our data shown above agree with their idea. If it is true, thepH gradient must also be generated by K' influx via a routeother than the Trkystem. As shown in Fig., the pH radientwas generated in the presence of valinomycin plus 50 mM K.The magnitude of the pH gradient generated under theseconditions is slightly higher than hat in the absence ofvalinomycin (Fig. 5). The identical data were reported withmembrane vesicles rom E. coli (33). In the presence ofvalinomycin, it is likely that the movement of K' is predom-inantly mediated by valinomycin. This result therefore showsthat K' influx via valinomycin lso supports the generation ofthe pH gradient.The generation of the pH gradient above 1.0 unit requiresa K concentration above 20 m~ in the presence of valino-mycin (Fig. 6), whereas the pH gradient generated in theabsence of valinomycin was reduced when the concentrationof K' was below 0.5 m~ (Fig. 6). In the absence of this drug,the pH gradient is generated by the aid of the Trksystem asmentioned above. It should be noted that theK , of the Trksystem is 0.5 m~ at pH 6.0 and this system is able to accu-mulate K until the external concentration of this cation fallsbelow 10 p~ (29). An interesting point shown in Fig. 6 is thatthe magnitude of the pH gradient generated in K+-depletedcells was only lightly higher than that in normal cells. Thus,

TABLEThe influx rate of K i s dependent on the eytoplasrniepH but noton the intracellular evel of KExperimental conditions and the data of the influx rate of K' arethe same as that in Fig. 4. Aliquots were filtrated at thearrow in Fig.4 and the intracellular concentration of K' (K+J was determined asdescribed under Materials and Methods. The cytoplasmic pH wasdetermined as described in the legend to Fig. 3. - ~ -.Experimental Influx rateconditions K+, pH,

pmol/mm/mg pmol/mgproteinproteinA 0.283 0.031

1.1 6.02.9C 7.3D 0.015 2.40.018 7.71.7 8.2

The value was obtained in the presence of 5 mM K' (maleate)instead of 2 mM KCI.

I I I I

10 20 30Tim min 1

FIG.5. Generation o the pH gradient in the presence ofvalinomycin.Washed cells of the wild type strain grown on mediumKTY were suspended in buffer (50 mM Tris/maleate, 2 m~ MgSO,,50 m~ K' (maleate), pH 6.0) at 0.40 mg of cell protein/&. The pHgradient was measured with acetylsalicylic acid in the presence 0 )and absence 0)f valinomycin (2 p g / m l as described underMaterials and Methods. Glucose (10 m ~ )as added at zero time.

I I 1 1 I I 1

K ( m M 1FIG. 6. Effect of the K + concentration on the generation ofthe pH gradient. Cells of the wild type strainwere grown on ediumKTY and washed with 2 m~ MgS0,. Internal K' was depleted asdescribed under Materials and Methods. Normal cells and depletedcells were suspended in buffer 50 m~ Tris/maleate, 2 mM MgSO,,pH 6.0). Various concentrations of K (maleate) and valinomycin (2pg/ml) were added to the cell suspension and the pH gradient wasmeasured with acetylsalicylic acid in the presence (closed symbols)

and absence (open symbols) of valinomycinas described under Ma-terials and Methods. Glucose 10 m ~ )as added at zero time.Sampling of aliquots was done at 10-18 min and 20-28 min in thepresence and absence of valinomycin, respectively. The experimentindicated as A was carried out using th e low concentration of cells toavoid the large change of the K' concentration in the medium.Symbols0 0 ormal cells;A, , depleted cells. Protein concentra-tions were 0.36 O), .40 O),0.34 A), nd 0.033 A)mg/ml.the magnitude of the pH gradient generated is dependent onthe concentration of extracellular K' and is independent ofthe initial concentration of intracellular K even if valinomy-cin is present.Mutant 7683, which is defective in the extrusion of Na', canaccumulate Na' (34) . This strainwas able to generate the pHgradient in the presence of Na', whereas no pH gradient wasgenerated in the wild type strain under the same condition(Fig. 7). It should be noted that thewild type strain is unableto accumulate Na+ under these conditions because of thefunction of the extrusion system for Na'. Mutant 7683 cangenerate the pH gradient of 1.3 units in the presence of K'(data not shown). Fig. 7 also shows that a small pH gradientis generated in the presence of 5 m~ Na' with the mutant7683, indicating that, if the influx of this cation is mediated bya carrier, the affinity of the carrier is very low. Heefner andHarold (34) indicated that Na' is accumulated by 7683 via arelatively nonspecific leak pathway for Na'. Thus, this resultindicates that the pH gradient can be generated even if acation flows into cells via a leak pathway.

The Cytoplasmicp H Is Regulated by the H+-ATPase t-self-we next examined why the influx rate of K decreasesas the cytoplasmic pH approaches 8.0 (Fig.4 and Table I). As


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13250 Regulation of BacterialytoplasmicHI EI I-

a 1.5-- .-A-A-A-A- -/ /-*-*-=: 1.0- -Cm-0.5

I -, o ~ - ~ - o - O - o -0 - - m1 m I . I90 10 2 0 0 1 2 0 30m -T h e { mln )FIG.7. Generationof the pH gradient in the presencef Na+.The wild type and 7683 strains were grown on medium KTY andintracellular K + was depleted as describedunder Materials andMethods. Depleted cells were suspended n buffer (50 mM Tris/maleate,2 MgS04, pH6.0)at 0.16 mg of cell protein/ml. The pHgradient was measured with acetylsalicylic acid as described underMaterials and Methods.he buffer contained the following cations:50 mnf Na (maleate) O), mnf Na (maleate) 0),nd 50 mnf K(maleate) A). , mutant 7683; B, wild type.mentioned above, an influx of K in S. faecalis is mediated bythe Trk system under the normalonditions (23,29). The Trksystem requires both ATP and the protonm otive force, andthe possibility that this sys tem is controlled by the proton -motive force has been proposed (29). There are two possibili-ties on this point. One is that the activityof the Trk systemis reduced a s the cytoplasmic pH reaches 8.0. The othe r isthat he protonm otive force generated by the H-ATPasedecreases and consequently theactivity of the Trk system isreduced. As shown in Table 11,a small pH gradient s gener-ated upon the addition of glucose at pH 8.0. The importantpoint is th at valinomycin has no essential effect on t he cyto-plasmic pH (Table 11).Cells of 7683 also generate a small pHgradient in th e presence of Na a t pH.0 (Table 11).Therefore,it is nlikely that the acti vityf th e H extrusion mediated bythe H-ATPase is still active but the cationnflux is repressedat pH 8.0.Abrams and Smith 35) have reported that theH-ATPaseof S. faecalis has a pHoptimum of th e AT P hydrolysisactivity at 6.0-8.0. We measured the activ ity in membranevesicles. A s shown in Fig. 8, the pH optimum of t he ATPhydrolysis activity is 6.0-7.0 and the activ itys very low at pHabove 8.0. This resul tsuggests that the acti vityf the protonextrusion mediated by th e H-ATPase is low at pH abo ve.0.The fact hat he generat ion of the protonmotive force ismarkedly reduced at pH above 7.5 (Fig. 1) also suggests thelow activity of the H+-ATPase t alkaline pH.

An another important point shown in Table I1 is that thecytoplasmic pH regulated n the presence of valinomycin plusK + is essentially identical with that regulated by the aid ofthe Trk system at ll external pH values tested. Thesimilarresult was obtained in muta nt 7683 in the presence of Nainstead of K, indicating that th e ytoplasmic pH is regula tedat near 8.0 even if the cation flow is mediated by a leakpathway. From these results, we conclude that the cytoplas-mic pH is regulated by the H+- ATPase itself but not by aspecific cation transport system, and the activi ty f alkaliza-tion of the cytoplasm decrea ses y the decline of the activ ityof the H-ATPase when thecytoplasmic pH approaches8.0.

Cells Also Alkalize the Cytoplasm at AlkalinepH-It asbeen proposed tha t, in E . coli and Bacillus alcalophilus, aninflux of proton mediated by sodium proton or potassiumproton antiporter regulates the cytoplasmic pH when thesebacteria are growing at alk aline pH 5 , 11-20). S. faecalis hasthe extrusion system for Na (34) and the presence of theextrusion system for K has been proposed (29). The cyto-plasm of S. faecalis is more acid than the medium in the

alkaline medium (Fig. I, Tables I1 and 111).In contrast to thehypothesis proposed with E . coli and B. alcalophilus, thecytoplasmic pH of S. faecalis is 7.6-7.9 in th e presence ofvarious ionophores in the medium of p H 8.4 (Table 111).Thisresult indic ates that the negative pH gradient (interior acid)of over 0.5 pH unit is generated when the generationof theprotonmotive force is blocked by ionophores. The same neg-ative gradient of pH wasobserved when ionophoreswereadded after the cytoplasm was regulated at pH 8.3 upon theaddition of glucose (da ta not shown). These results suggestth at cells alkalize th e cytoplasm from pH near 7.7 to p H 8.3by the aid of the H+-AT Pase.

The abilityof AT P production by the arginine metabolismis lower than that by glycolysis. Th e pH values of the cyt o-plasm were 6.7 and 7.3 in the mediumf pH 6.0 when arginineand glucose, respectively, were added. Thus, if cells acidifythe cytoplasm in the alkaline medium, its expected that thenegative pH gradient (interior acid) generatedy the additionof arginine is smaller than that generated by the addit ion ofglucose. However, the cytoplasm attained upon the additionof arginine is more acid than that atta ined pon the additio nof glucose in the medium of pH 8.4 (Tab le I I I , supportingthat cells alkalize the cytoplasm in the alkal ine medium.It is possible that the cytopla sm iscidified by the produc-tion of lacta te during lycolysis. However, the cytoplasmic pHwas 8.0 when arginine was added instead of glucose in the

TABLE1Effects of cations and ionophores on the cytoplasmic pH

The cytoplasmic pHwas determined as described in the legend toFig. 1 except that methylamine was used at pH 8.6. Washed cells ofthe wild type strain grown on medium KTYwere suspended in buffercontaining 50 mM K (maleate) at 0.30-0.40 mg of cell protein/ml.Valinomycin was added at 2 pg/ml. The cytoplasmic pH in mutant7683 was measured as described above except that K+-depletedcellsprepared as described under Materials and Methods were used and50 mM Na+ (maleate) was used instead of K. Protein concentrationwas 0.15-0.20 mdml.

pH, . ,Wild K 7.3 8.0 8.2 8.4Wild K + 7.5 8.0 8.27683 Na 7.3.8.1.3 8.4

A

I I I8 7 8 9pH

FIG. 8. Activity of ATP hydrolysis by the H+-ATPase as afunction of the pH of the medium in membrane vesicles fromthe wild type strain. Membrane vesicles were prepared from thewild type strain as described under Materials and Methods andsuspended in buffer at 0.08mg of protein/ml. The activity of ATPhydrolysis was assayedas described under Materials ane Methods.The activity represents micromoles of inorganic phosphate formedper min per mg of protein. Symbols:A, 0 Tris/maleate buffer;0,90mM Tris/Tricine buffer.


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Regulation of Bacterial Cytoplasmic H 13251TABLE11

Cytoplasmic pH in the presence of ionophoresWashed cells of the wild type strain grown on medium K T Y weresuspended in the buffer containing 50 mM Tris/Tricine, 2 mM MgSO,,and 50 mM K maleate),pH 8.4, at 0.34-0.37 mg of cell protein/ml.Cells adapted to arginine were grown on the medium, pH 8.5, con-taining arginine instead of glucose as described under Materials andMethods. TCS (10PM , alinomycin ( 2 pg/ml), nigericin ( 2 pg/ml),

and gramicidin D 4 pg/ml) were added to the cell suspension. TenmM glucose or arginine was added at zero time and the cytoplasmicpH was determined with methylamine at 15-25 min as describedunder Materials and Methods. __nergy sourceGlucose ArginineIonophores

pHrr ,None 8.3 8 0TCS 7.7 7.7Valinomycin plus nigericin 7.9 7.8Gramicidin D 7.6 7.6medium of pH 8.4 (Table 111). It is also shown in Table I11that, in the presence of ionophores, the cytoplasmic pH at-tained upon the addition of arginine is essentially identicalwith that atta ined pon the addition of glucose. These resultsindicate that thenegative pH gradient is not generated by theproduction of lactate because lactate is not produced duringthe arginine metabolism. We also measured the cytoplasmicpH of cells incubated with ionophores in the absence of theenergy source and the results were essentially identical withthat shown in Table 111. The negative pH gradient of thesimilar magnitude was also observed in the presence of iono-phores a t pH 6.0 (data not shown). These results suggest tha tthe generation of the negative pH gradient is not dependenton the energy metabolism. Thus, this negative gradient of pHseems to be generated by some physical force, for exampleDonnan potential.From these results, we conclude that 1) cells alkalize thecytoplasm with the aid of the H-ATPase at all pH valuesand 2) a t alkaline pH, the cytoplasmic pH cannot exceed themedium pH because of the low activity of the alkalization.Finally, one might point out tha t the pH gradient generatedat pH above 8.0 is too small to argue. However, as mentionedunder Materials and Methods, an error of the values pre-sented in Tables I1 and I11 is less than 0.1 pH unit.

DISCUSSIONWe propose, in this paper, he mechanism for the regulationof the cytoplasmic pH in Streptococcus faecalis.Th e essential

points are thefollowing: 1) this bacterium has a machinery toraise the cytoplasmic pH butnot one to lower it, 2) thecytoplasmic pH is raised by the extrusion of protons mediatedby the H-ATPase and theelectrogenic influx of cations, and3) when the cytoplasmic pH approaches 8.0, the activity ofthe alkalization decreases by the decline of the activity of theH-ATPase and, consequently, the cytoplasmic pH is kept atnear 8.0.In our model, the magnitude of the tota l rotonmotive forceis dependent on the activity of the H-ATPase and cellsmaximize the pHgradient.Here, it remains unclear whatdetermines the maximum magnitude of the pHgradient.When cations flow into cells, the magnitude of the membranepotential decreases and the magnitude of the pH gradientincreases (8, 29, 30). We think now t hat the influx rate ofcations determines he magnitude of the membrane potential.When the influx rate of cations is high, the magnitude of themembrane potential is small and the magnitude of the pHgradient is large. As discussed above, K + influx via the Trksystem is faster than Na influx via t,he leak pathway. Th e

rate of K influx via valinomycin is thought to be higher thantha t via the Trk system if the sufficient amount of K ispresent. The magnitudes of the pH gradient generated corre-spond well with the influx rates of cations (Figs.5 and 7 ) .

Our data suggest that the cytoplasmic pH is controlled bythe pH dependence of the H-ATPase activity although cationflux is required for the generation of the pH gradient. Thismeans that the primary function of the H+-ATPase is theregulation of the cytoplasmic pH. Raven and Smith (36) haveargued tha t the primitive function of a H+-ATPase is anextrusion of protons for the regulation of the internal pH. Itis very interesting that their argument applies to one of themodern bacteria, S. faecalis, if our model is true.

It has been proposed tha t anefflux of protons mediated bylactate efflux can generate the protonmotive force 37-39).Protons are eally expelled in AS25when glucose is added andthis efflux is not inhibited by DCCD (21). We suppose thatthis efflux is mediated by the efflux of lactate. However, ourdata (21, this paper) indicate that the proton efflux mediatedby the lactate efflux is involved in neither the generation ofthe protonmotive force nor the alkalization of the cytoplasmunder our conditions. So far, the true reason for this conflictis unknown. It is quite conceivable that the protonmotiveforce canbe generated only when the high concentrationgradient of lactate is established artificiaily.S. faecalis has a leak pathway for Na and Ca2 (34, 22).The TrkF ystem of E . coli is probably a leak pathway or Kbecause this system has a very high K , ( S O 0 mM) and themutant defective in this system has never been isolated (40).It is still unclear whether S. faecalis has such a pathway forK+ or not, although the presence of such a pathway has beenproposed 29). If S. faecalis has sucha pathway, the pHgradient can be generated with K influx via the pathway.The rate f K accumulation via the leak pathway is probablylow. The affinity of such a pathway for K is probably verylow. Consequently, the generation of the pHgradient accorn-plished with K influx via the leak pathway is thought to beslow and o equire the high concentration of K in themedium. In fact, themagnitude of the pH gradient generatedwith the influx of Na is small and requires the high concen-tration of Na as compared with that accomplished with Kinflux via the Trk system (Fig. 7 ) .Thus, we suppose that S.faecalis uses the Trk system to regulate the cytoplasmic pHunder normal conditions.Our data suggest tha t an electrogenic influx of K+ via theTrk system rather than a net uptake is important for theregulation of the cytoplasmic pH. The Trk system mediatesunidirectional accumulation of K+ andequires both ATP andthe protonmotive force (29). Two models for this transportsystem have een proposed (29).One isthe ATP-driven pump,regulated by t he protonmotive force, and the other is thesystem linked to proton circulation, regulated by ATP. Aspresented in his paper, the influxaf K declines as heactivity of H+-ATPase declines when the cytoplasmic pHreaches 8.0. Therefore, the former model for the Trk systemfits our model. Kroll and Booth (9) have reported that netuptake of K led to the generation of a pH gradient in K-depleted cells but the H gradient s generated in K+-replacedcells although no net uptake ofK occurs in E . coli. Theyhave proposed that a cycling of K rather than a net uptakeis important for the regulation of the cytoplasmic pH (9).A s suggested here, K is accumulated for th e regulation ofthe cytoplasmic pH. On the otherhand, cells must control helevel of cellular K+. It is possible that the nternal level of thiscation is controlled by the efflux system althoughwe have nodirect evidence. Bakker and Harold (29) have proposed thatan efflux system is different from the influx system for K


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13252 Regulation of Bacterial CytoplasmicpH(29). S. faecalis has a second transport system for K + accu-mulation, theKdpsystem,and t is expressed when hecellular level of K+ is low (23). Thus, it is also likely that K'is accumulated y the Kdp system when theufficient amountof K' is not accumulateds he result f the regulation f thecytoplasmic pH. The study on his matter is in progress.Considerable research has been done on the regulation ofthe cytoplasmic pH in E . coli (for review, see Ref. 5). It hasbeen pointed out tha t theytoplasmic pH is regulated by thegenerator of the protonmotive orce and the transport systemfor K+ at pH below 7.5 8, ). An important point observedpreviously is t hat the internal pH is also kept constant inmembrane vesicles from E . coli (33) and, to my best knowl-edge, there has been no report to document theK accumu-lation in membrane vesicles. Inour model, cation flow isessentialbut ts oute is not mportant. It is likely thatmembrane vesicles are somewhat permeable for cations venthough they have no transport systemor K+.

Several reports havepointed out that sodium proton anti-porter or potassium proton antiporter regulates the cytoplas-mic pH when cells of E . coli are growing at pH above8.0 (5 ,11-13, 15-17). However, it is also pointed out tha t the cyto -plasmic pH is regulated without the additionf Na' or K' ( 5 ,41), and a mutant defective in potassium proton antiporterregulates the ytoplasmic pH normally (41). In ourmodel, thecytoplasmic pH s lower than he medium pHwhen healkalization of the cytoplasm is blocked or its activityis low.Th e lower pH of the cytoplasm in the absence of Na' or K +is not so puzzling in our model. So far, there has been noobservation to suggest that E . coli has a similarsystem to thatof S. faecalis but we would state one observation that theactivity of respiration is markedly decreased a t pH bove 8.011).Finally, let u s now turn to the very importan t point inbioenergetics. A s shown here, thecytoplasm is more acid than

the medium when the machinery to r aise thecytoplasm pHdoes not function. Bakker and Mangerich (8) have reportedthat, in S. faecalis, the pH gradient isegative in he presenceof the low concentration of K + , supporting our data. Severaldata suggest that this cid pH of the cytoplasms independentfrom the energy metabolism. Thus, the most likely explana-tion is that thisnegative pH gradient is generatedy Donnanpotential. This may e supported by the fact that this negativegradient is decreased by the increase of K' Concentration.' Ifour argument s true, the term f Donnan potential should eadded to the protonmotive orce.

Acknowledgments-We are indebted to Dr. Franklin M. Haroldfor the critical reading of the manuscript and his invaluable sugges-tions.

REFERENCES1. Mitchell. P. (1976) Biochem. Soc. Trans. 4,399-4302. Harold, F. M. (1977) Curr. Top. Bioenerg. 6,83-1493. Harold, F. M., and Van Brunt, J. (1977) Science 19 7, 372-373

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4. Harold, F. M. (1977) Annu. Reu. Microbiol. 31, 181-2035. Padan, E., Zilberstein, D., and Schuldiner, S. (1981) Biochim.6. Harold, F. M., Pavlasova, E., and Baarda, J. R . (1970) Biochim.7. Harold, F. M., and Altendorf, K. (1974) Curr. Top. Membr. Trans.8. Bakker, E. P., and Mangerich, W. E. (1981) J. Bucteriol. 147,9. Kroll, R. G., and Booth, I. R. (1981) Biochem. J . 198, 691-698

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